Growth/differentiation factor of the TGF-beta family

ABSTRACT

The invention relates to an antibody or antibody fragment which specifically binds to a protein of the TGF-β family. The invention also relates to a kit for detecting a protein of the TGF-β family which comprises the antibody or antibody fragment. Finally, the invention relates to method for detecting a protein of the TGF-β family which uses the antibody or antibody fragment.

The present invention concerns a new growth/differentiation factor ofthe TGF-β family and DNA sequences coding therefor.

The BMP-, TGF- and inhibin-related proteins are members of the TOP-βfamily of growth factors (Roberts and Sporn, Handbook of ExperimentalPharmacology 95, 419-472 (1990)). They are relevant for a wide range ofmedical therapeutic methods and applications. These factors are suitablefor methods relating to wound healing and tissue regeneration. Moreoverseveral members of the TGF-β family induce tissue growth for example thegrowth of bones.

Wozney (Progress in Growth Factor Research 1 (1989), 267-280) and Valeet al. (handbook of Experimental Pharmacology 95 (1990), 211-248)describe various growth factors for example those which are related tothe BMP and the activin/inhibin group. The members of this group havesignificant structural similarities. The precursor of the protein iscomposed of an amino-terminal signal sequence, a propeptide sequence anda carboxy-terminal sequence of 110 to 140 amino acids which is cleavedfrom the precursor and represents the mature protein. Furthermore itsmembers are defined by an amino acid sequence homology. The matureprotein contains the sequences that are conserved most, in particularseven cysteine residues which are conserved among the family members.The TGF-β-like proteins are multifunctional, hormonally active growthfactors. They oleo have related biological activities for examplechemotactic attraction of cells, promotion of cell differentiation andtissue-inducing capabilities. EP 0 222 491 A1 discloses sequences ofinhibin alpha and beta chains.

On the whole the proteins of the TGF-β family show differences in theirstructure which leads to considerable variations in their exactbiological function. In addition they are found in a wide range ofdifferent types of tissues and stages of development. As a consequencethey may be different with regard to their exact junction e.g. therequired cellular physiological environment, their life span, theirtarget areas, their requirements for auxiliary factors and theirresistance to degradation. Although numerous proteins that showtissue-inductive potential have been described, their natural functionsin the organism and—even more importantly—their medical relevance stillhas to be researched in detail. It can in all probability be assumedthat there are still unknown members of the TGF-β family which are ofimportance for the differentiation/induction of various types of tissue.However, a major difficulty in the isolation of these new TGF-β-likeproteins is that their functions cannot yet be described preciselyenough to develop a highly discriminating bioassay. On the other handthe expected nucleotide sequence homology to known members of the familyin too small to enable screening by classical nucleic acid hybridizationtechniques. Nevertheless the further isolation and characterization ofnew TGF-β-like proteins is urgently required in order to provide furtherinducing and differentiation proteins which fulfil all medicalrequirements. These factors could be used medically in healing injuriesand treating degenerative diseases of various tissues.

A nucleotide and amino acid sequence for the TGF-β protein MP121 isgiven in the patent application PCT/EP93/00350 in which a major part ofthe sequence corresponding to the mature protein is stated. The completesequence of the propeptide MP121 is not disclosed.

The underlying object of the present invention in to provide DNAsequences which code for new members of the TGF-β protein family withmitogenic and/or differentiation-inductive potential. The object of thepresent invention is in particular to provide the complete DNA and aminoacid sequence of the TGF protein MP121.

This object is achieved by a DNA molecule that codes for a protein ofthe TGF-β family and which comprises:

-   (a) the part coding for the mature protein and if necessary further    functional parts of the nucleotide sequence shown in SEQ ID NO. 1,-   (b) a nucleotide sequence corresponding to the sequence from (a)    within the scope of the degeneracy of the genetic code,-   (c) a nucleotide sequence corresponding to an allelic derivative of    one of the sequences from (a) and (b) or-   (d) a sequence which differs from sequence (a) due to the fact that    it originates from other vertebrates-   (e) a sequence hybridizing with one of the sequences from (a),    (b), (c) or (d)-   provided that a DNA molecule according to (e) contains at least the    part coding for a mature protein of the TGF-β family.

Further embodiments of the present invention concern the subject matterof claims 2 to 10. Other features and advantages of the invention emergefrom the description of the preferred embodiments. The sequenceprotocols and drawings are now briefly described.

SEQ. ID NO. 1 shows the complete nucleotide sequence of the DNA codingfor rile human TGF-β protein MP121. The ATG start codon begins atnucleotide 128. The start of the complete mature protein particularlypreferably begins at nucleotide 836.

SEQ ID NO. 2 shows the complete amino acid sequence of the preproproteinof the human TGF-β protein MP121 which was derived from the nucleotidesequence shown in SEQ ID NO. 1. The start of the nature protein ispreferably in the region of amino acids 217-240, particularly preferablyat amino acid 236 or 237 and most preferably at amino acid 237.

SEQ ID NO.3 shows the complete nucleotide sequence of the DNA coding forthe TGF-β protein MP121 from the mouse. The coding region begins at theATG start codon at nucleotide 131 and ends at the stop codon beginningat position 1187. The start of the mature protein preferably begins atnucleotide 839. A ca. 5.5 kb large intron is located in the genomic DNAbetween position 446 and 447.

SEQ ID NO. 4 shows the complete amino acid sequence of the preproproteinof the TCP-β protein MP121 from the mouse which has been derived fromthe nucleotide sequence shown in SEQ ID NO. 3. The mature protein beginsin the region of amino acids 217-240 in analogy to the human MP121 ofSEQ ID NO.2. It is most preferred when the mature protein starts atamino acid 237 so that the mature part consists of 116 amino acids as inthe human MP121. Members of the TGF-β family are frequently cleavedbehind a RXXR cleavage site in order to separate the mature part fromthe precursor (see Ozkaynak et al., J. Biol. Chem. 267, 25220-25227(1992) and the literature cited therein). In the case of MP121 from themouse it is also conceivable that the beginning of the mature protein isat least sometimes at amino acid 236.

SEQ ID NO.5 shows the nucleotide sequence of the human MP121 gene at theexon/intron junctions. The nucleotides from both exons are marked bycapital letters those of the intron by small letters.

FIG. 1 shows a comparison of the amino acid sequence of human MP121 withsome members of the TGF-β family (inhibin α and β chains) starting atthe first of the seven conserved cysteine residues. * denotes that theamino acid is the same in all compared proteins; + denotes that theamino acid corresponds in at least one of the proteins compared to humanMP121.

FIG. 2 shows the nucleotide sequences of the oligonucleotide primerswhich were used in the present invention and a comparison of thesesequences with known members of the TGF-β family. M denotes A or C, 9denotes C or G, R denotes A or G and K denotes G or T. 2 a shows thesequence of primer OD, 2 b shows the sequence of primer OID.

FIG. 3 shows a diagram of a Western blot using chicken antibodiesagainst human MP121.

FIG. 4 shows the expression of MP121 compared to activin β_(A) and β_(B)in various mouse tissues.

FIG. 5 shows a positive influence on the survival of dopaminergicneurones by treatment with partially purified MP121.

FIG. 6 shows a Western blot using rabbit antibodies against human MP121.

FIG. 7 shows the stimulation of nerve fibre outgrowth from the embryonicretina by treatment with partially purified MP121.

FIG. 8 shows that partially purified MP121 can inhibit EGF induced DNAsynthesis in hepatocytes.

FIG. 9 shows the influence of partially purified MP121 on erythroiddifferentiation.

Within the scope of the present invention the term “mature protein” alsoencompasses functional partial regions of the complete protein whichexhibit essentially the same biological activity and preferably thosepartial regions which include at least the region of the seven cysteinesthat are conserved in the TGF-β family. In this case it is in particularpossible that the N-terminus of the mature protein is slightly modifiedi.e. deviates from the sequences shown in SEQ ID NO.2 and 4. In thisconnection additional amino acids, which do not influence thefunctionality of the protein, may be present or amino acids may beabsent provided that in this case the functionality is also notimpaired. However, it is preferred that the human protein and the mouseprotein contain all amino acids starting with amino acid 237 of theamino acid sequence shown in SEQ ID NO.2 and SEQ ID NO.4. It is alreadyknown from other family members of the TGF-β family that the attachmentof additional amino acids to the N-terminus of the mature protein doesnot influence the activity wherein inter alia 6 additional histidineswere attached to the N-terminus.

Therefore the present invention encompasses the part coding for themature protein in accordance with the above-mentioned definition and ifnecessary, further functional parts of the nucleotide sequence shown inSEQ ID NO. 1 as well as sequences that correspond to this sequencewithin the scope of the degeneracy of the genetic code and allelicderivatives of such sequences. Furthermore the present invention alsoencompasses DNA sequences which code for a protein of the TGF-β familywhich were obtained from other mammals and which have a sequence thatdeviates slightly due to their origin but which, however, code forproteins having in principle the same biological function and alsosequences that differ only slightly. Such sequences correspond to oneanother to a very large extent as can be seen by comparing SEQ ID NO. 1and NO. 3.

In addition the present invention also covers sequences hybridizing withsuch sequences provided that such a DNA molecule at least completelycontains the part coding for a mature protein of the TGF-β family(according to the above definition) and the biological activity isretained.

The term “functional part” within the sense of the present inventiondenotes a protein part which is capable of acting for example as asignal peptide, propeptide or mature protein moiety i.e. it fulfills atleast one of the biological functions of the natural parts of MP121.

In the case of the preferred human MP121 the region coding for themature part of the protein preferably extends from nucleotide 836 to thestop codon which begins at nucleotide 1184 of the sequence shown in SEQID NO. 1. If necessary, the DNA molecule can include further functionalparts of the sequence shown in SEQ ID NO. 1 namely the nucleotidesequences coding for the signal or/and propeptide part. It inparticularly preferred that the DNA molecule comprises the sequence forthe signal and propeptide part and the mature protein part i.e.nucleotides 128 to 1184 of the sequence shown in SEQ ID NO. 1 In thecase of the preferred mouse MP121 the region coding for the mature partof the protein preferably extends from nucleotide 839 to the stop codonstarting at position 1187 of the sequence shown in SEQ ID NO.3. Ifdesired the DNA molecule can also include further functional parts ofthe sequence shown in SEQ ID NO.3 i.e. if desired nucleotide sequencescoding for the signal or/and propeptide part.

On the other hand the DNA molecules can also include functional signalor/and propeptide parts of other proteins e.g. of proteins with thecystine knot motif (Cell, vol. 73 (1993) p. 421-424) and in particularof other proteins of the TGF-β family e.g. the abovementionedactivin/inhibin or BMP proteins especially also MP52 (seePCT/EP94/02630) in addition to the part coding for the mature protein.The respective nucleotide sequences can be found in the aforementionedreferences to the disclosure of which reference is herewith made. Inthis case it is important that the correct reading frame for the matureprotein is preserved. Depending in which host cells expression takesplace, the presence of another signal sequence or/and of anotherpropeptide part may positively influence the expression. The exchange ofpropeptide parts by corresponding parts of other proteins is describedfor example in Mol. Endocrinol. 5 (1991), 149-155 and Proc. Natl. Acad.Sci. USA 90 (1993), 2905-2909.

Although the allelic, degenerated and hybridizing sequences andsequences derived from other vertebrates which are covered by thepresent invention have structural differences due to slight changes inthe nucleotide or/and amino acid sequence, proteins which are coded byouch sequences still essentially have the same useful properties whichenable them to be used in essentially the same medical fields ofapplication.

According to the present invention the term “hybridization” denotes theusual hybridization conditions, preferably conditions with a saltconcentration of 6×SSC at 62 to 66° C. followed by a one hour wash with0.6×SSC, 0.1% SDS at 62 to 66° C.

Preferred embodiments of the present invention are DNA sequences asdefined above which are obtainable from vertebrates, preferably mammalssuch as pigs, cows and rodents such as rats or mice and in particularfrom primates such as humans or which are copied from correspondingsequences.

A particularly preferred embodiment of the present invention are thesequences shown in SEQ ID NO. 1 and 3 and denoted human or mouse MP121sequences. The transcripts of MP121 were obtained from liver tissue andcode for a protein which shows a considerable amino acid homology to themature part of the inhibin/activin-like proteins (see FIG. 1). Theprotein sequences of human α-inhibin, inhibin β_(A), (activin β_(A)) andinhibin β_(B) (activin β_(B)) are described by Mason et al. (Biochem.Biophys. Res. Comm. 135, 957-964 (1986)). Some typical sequencehomologies which are specific for known inhibin sequences were alsofound in the propeptide part of MP121 while other parts of thepropeptide of MP121 show considerable differences to inhibinpropeptides.

However previous findings show that there are differences between thepattern of expression of MP121 and that of the activins. While activinsare mainly expressed in the gonads (activin β_(A) in ovaries and activinβ_(B) in testes and ovaries), MP121 is mainly expressed in the liver.However up to now the sensitivity of the experiments has not beensufficient to also detect a slight expression. Thus in the case ofactivins it has for example been described in the literature thatexpression can also be detected outside the gonads in various rattissues in adult animals (Meunier et al., Proc. Natl. Acad. Sci. USA 85,247-251 (1980)) an well as during embryonic development (Roberts et al.,Endocrinology 128, 3122-3129 (1991)). Therefore it is also possible thatexpression of MP121 in other tissues may yet be detected.

Because of the predominant expression of MP121 in liver the expressionin one typical cell type of the liver was investigated in more detail.It was shown that the mRNA is expressed abundantly in cultured primaryrat hepatocytes as well as in liver cell lines such as HepG2 (ATCC Hβ8065). The expression in primary cells is markedly reduced by EGF(Epidermal Growth Factor) treatment after 60 hours. This pattern iscompletely different compared to activin βA mRNA, which is barelyexpressed in hepatocytes but increased drastically after EGF treatment(Yasuda et al., J. Clin. Invest. Vol. 92, 1491-1496 (1993)). Likewise,the expression of activin βA in mRNA and MP121 mRNA is reciprocal inremnant rat liver after 70% hepatectomy. MP121 mRNA is detectedsignificantly before hepatectomy but is markedly decreased after 12hours or later, whereas the mRNA for activin βA is quite low before butelevated 12 hours or later after hepatectomy. Therefore MP121 seems tohave a big influence on the ability of the liver to regenerate andproliferate. The control of MP121 mRNA expression and/or the amount ofMP121 protein in liver can be of significance for treatment of livercarcinomas, liver injuries or diseases such as for example cirrhoticliver.

In addition the present invention concerns a vector which contains atleast one copy of a DNA molecule according to the invention. In ouch avector the DNA sequence according to the invention is preferably linkedoperatively with an expression control sequence. Such vectors aresuitable for producing TGF-β-like proteins in stably ortransiently-transformed cells. Various animal, plant, fungal andbacterial systems can be used for the transformation and the subsequentculture. The vectors according to the invention preferably containsequences necessary for replication in the host cell and they areautonomously replicable. In addition the use of vectors is preferredwhich contain selectable marker genes by which means the transformationof a host cell can be detected.

Furthermore the invention concerns a host cell which is transformed witha DNA according to the invention or with a vector according to theinvention. Examples of suitable host cells include various eukaryoticand prokaryotic cells such as E. coli, insect cells, plant cells,mammalian cells and fungi such as yeast.

In addition the invention concerns a protein of the TGF-β family whichis coded by a DNA sequence according to claim 1. The protein accordingto the invention preferably has the amino acid sequence shown in SEQ IDNO. 2 or in SEQ ID NO.4 or if desired functional parts thereof (asdefined above) and exhibits biological properties such astissue-inductive properties which may be relevant for a therapeuticapplication. The above-mentioned features of the protein can varydepending on the formation of homodimers or heterodimers with otherproteins having the “cystine knot motif” and in particular TGF-βproteins. Such structures may also prove to be suitable for clinicalapplications and thus are also a subject matter of the presentinvention. Preferred heterodimers include heterodimers composed of amonomer of the protein according to the invention and monomers of the α,β_(A) or β_(B) inhibin chains. The properties resulting from heterodimerformation can be shifted more towards the properties of activin orinhibins. If for example a heterodimer is formed with inhibin a proteinsor with other inhibin β proteins, then it is assumed that theMP121/inhibin (α chain) or MP 121/activin (β_(A) or β_(B) chain)heterodimer can inhibit or activate the formation offollicle-stimulating hormone (FSH). MP121/activin heterodimers may alsofor example influence mesoderm development. Furthermore it is expectedthat heterodimeric forms with a member of the BMP group of TGF-βproteins lead to an amplification of BMP-like activities such as forexample the ability to induce or promote bone formation, formation ofcartilage or formation of connective tissue.

The invention therefore also concerns heterodimeric proteins of aprotein of the TGF-β family according to the invention which is coded bya DNA sequence as claimed in claim 1 containing a monomer of a proteinwith the “cystine knot motif” preferably of another member of the TGF-βfamily. Similar heterodimeric proteins are described in WO93/09229, EP 0626 451 A2 and J. Biol. Chem. 265 (1990), 13198-13205.

In addition the invention concerns chimeric proteins which havefunctional derivatives or parts of a protein coded by a DNA sequenceaccording to the invention preferably as shown in SEQ ID NO.2 or SEQ IDNO.4, in particular functional parts of the mature protein andadditionally parts of another protein. In this case the other proteincan also be a protein with a “cystine knot motif” which is preferablyalso a member of the TGF-β family such as e.g. especially MP52(PCT/EP94/02630). However, parts of a complete different protein canalso be present e.g. receptor-binding domains of proteins which lend theinitial MP121 protein another specificity.

The biological properties of the proteins according to the invention,preferably MP121, can be determined for example in assays according toWrana et al., (Cell 71, 1003-1014 (1992)), Ling et al. (Proc. Natl.Acad. of Science, 82, 7217-7221 (1985)), Takuwa et al. (Am. J. Physiol.257, E797-E803 (1989)), Pann and Patterson (Proc. Natl. Acad. ofScience, 91, 43-47 (1994)), Broxmeyer et al. (Proc. Natl. Acad. ofScience, 85, 9052-9056 (1988)), Green et al. (Cell, 71, 731-739 (1992))or Partridge et al. (Endocrinology, 108, 213-219 (1981)) or Krieglsteinet al. (EMBO J. 14, 736-742 (1995)).

Activin A and TGF-β 1, TGF-β 2 and TGF-β 3 have been described topromote survival of dopaminergic neurones in vitro (Krieglstein et al.,EMBO J. 14, 736-742 (1995) and Krieglstein et al., Neuroscience 63,1189-1196 (1994)). In the case of partially purified MP121 it could beshown that the survival of dopaminergic neurones in a B-day culture ispromoted to a greater extent than by the influence of the controlsupernatant (FIG. 5).

During the development of the visual system a projection of axons fromthe retinal ganglion cells to the special regions in the brain inestablished. It was shown by several groups that soluble factorsisolated from visual areas of the brain can trophically stimulateretinal ganglion cells (Nurcombe, V. & Bennett, M. R., Exp. Brain Res.44, 249-258 (1981), Hyndman, A. G., Adler, R., Dev. Neurosci. 5, 40-53(1902), Turner, J. E. et al., Dev. Brain Res. 6, 77-83 (1903), Carri, N.G. & Ebendal, T., Dev. Brain Res. 6, 219-229 (1983)). The formation ofnerve fibre fascicles, which most likely represent optic axons stemmingfrom the retinal ganglion cells, depends on neurotrophic factors. UsingMP121, a strong stimulation of retinal nerve fibre outgrowth in explantcultures of the embryonic chicken retina was evident as shown in Tab.1and FIG. 7. During these experiments, other members of the TGF-βsuperfamily, as for example MP52 (DE, 195 25 416.3), were also proven tobe active.

This activity of MP121 can be useful for the treatment of diseases atthe eye as for example the retina or the optic nerve. It is especiallyuseful for injuries of the neural retina and the optic nerve. Suchinjuries can be evoked for example by accidents, inflammations ordisturbance of the supply of blood. It can also be useful for thetransplantation of the retina. Furthermore the treatment of othercerebral nerves is important. One example is the trigeminal nerve(Nervus trigeminus), which also provides parts of the eye. Therefore,members of the TGF-β family, especially MP52 and MP121, can also beuseful for the transplantation of the cornea. Additionally the treatmentof partial damage of the cornea as for example evoked by a herpesinfection can be possible. Furthermore the treatment of degenerativedisorders at the surface of the eye are of interest.

Results on rat hepatocytes in primary cultures indicate that partialpurified MP121 inhibits the initiation of DNA synthesis (FIG. 8). Theeffect of MP121 resembles that of Activin A and TGF-β (Yasuda et al., J.Clin. Invest, Vol. 92, 1491-1496 (1993)) but the concentrations of MP121which are necessary to block the growth promoting actions of EGF arehigher. Nevertheless it can be assumed that MP121 can influence livergrowth. Therefore it can be useful in several liver diseases includingliver carcinomas.

Activin A is furthermore known or its ability to promote thedifferentiation of Friend erythroleukemic cells (F5-5) wherefore it wasalso designated Erythroid differentiation factor (EDF) (Eto et al.,Biochem. Biophys. Res. Com. 142, 1095-1103 (1987)). Partial purifiedMP121 shows also a slight activity in this assay system. Therefore MP121can be useful in stimulation of erythropoiesis.

The present invention in addition concerns a process for the productionof a protein of the TGF-β family which is characterized in that a hostcell transformed with a DNA according to the invention or with a vectoraccording to the invention is cultured and the TGF-β protein is isolatedfrown the cell or/and the culture supernatant. Such a process comprisesculturing the transformed host cell in a suitable culture medium andpurifying the TGF-β-like protein formed. In this way the process enablesthe production of an adequate amount of the desired protein for use inmedical treatment or in applications using cell culture techniques inwhich growth factors are needed. The host cell can be a bacterium suchas Bacillus or E. coli, a fungi such as yeast, a plant cell such astobacco, potato or arabidopsis or an animal cell, especially avertebrate animal cell line such as Mo, Cos or CHO cell lines or aninsect cell line. Using the Baculovirus system, expression can also beperformed in insect larvae. When producing in bacteria it is possiblethat the protein according to the invention is produced in the form ofinclusion bodies. These inclusion bodies are then renatured according toknown methods and the protein is then obtained in an active form (seee.g. Jaenicke, R. and Rudolph, R., Protein Structure, ed. Creighton, T.E. IRL Press, chapter 9). For the production of heterodimeric proteinswith other members of the TGF-β family, both protein monomers areexpressed either in the same cell or separate in the course of which acommon renaturation seems suitable with formation of inclusion bodies.Viral systems such as e.g. the Baculovirus system or the Vaccina virussystem are in particular suitable when coexpressing in the same cell.The production of heterodimeric proteins is in principle known to aperson skilled in the art and is described for example WO93/09229 and BP0 626 451 A2.

The production of chimeric proteins containing other protein partsrequires a corresponding change at the DNA level which is familiar to aperson skilled in the art and can be carried out by him (EMBO J. 10(1991), 2105-2110; Cell 69 (1992), 329-341, J. Neurosci. 39 (1994),195-210).

Yet another subject matter of the present invention is the provision ofpharmaceutical compositions which contain a pharmaceutically effectiveamount of a TGF-β-like protein according to the invention as the activesubstance. If desired, such a composition comprises a pharmaceuticallyacceptable carrier or auxiliary substance, diluent or filling agent.Such a pharmaceutical composition can be used alone or in combinationwith other active substances for example other proteins of the TGF-βfamily or growth factors such as EGF (epidermal growth factor) or PDGF(platelet derived growth factor) in wound healing and tissueregeneration. Furthermore such a pharmaceutical composition can be usedfor the prevention of diseases.

Further subject matters are pharmaceutical compositions which containheterodimeric proteins or/and chimeric proteins according to theinvention.

The pharmaceutical composition according to the invention is preferablyused for the treatment and prevention of damage to bones, cartilage,liver, connective tissue, skin, mucous membranes, endothelium,epithelium, neurones, kidneys or teeth, for application in dentalimplants, for application in wound healing or tissue regenerationprocesses, induction of the proliferation of precursor cells or bonemarrow cells, for the maintenance of a state of differentiation and forthe treatment of disturbances in fertility or for contraception.

Furthermore the pharmaceutical composition according to the inventioncan be useful for the treatment of diseases concerning the metabolism,such as digestive disorders or disorders concerning the level ofbloodsugar.

A further possible clinical application of the TGF-β-like proteinaccording to the invention is the use as a suppressor of immunoreactionsin order to avoid rejection of organ transplants or use in connectionwith angiogenesis.

Furthermore the protein according to the invention can be used toincrease fertility or in contraception. The pharmaceutical compositionaccording to the invention can also be used prophylactically or incosmetic surgery. Furthermore the application of the compositions is notlimited to humans but can also include animals in particular pets anddomestic animals.

Thus the part of the other protein or other monomer can be used to varythe scope of applications and specificity of heterodimeric proteins andchimeric proteins as desired.

In general diseases which are associated with the expression of MP121can be treated using the proteins according to the invention either byincreasing the amount or activity of MP121 which is present or bysuppressing the MP121 activity. Thus the invention also concerns theproduction of antisense nucleic acids and ribozymes which inhibit thetranslation of MP121. This inhibition can either be achieved by maskingthe mRNA with an antisense nucleic acid or by cleavage with a ribozyme.

The production of antisense nucleic acids is known (Weintraub, H. M.,Scientific American 262: 40 (1990)). The antisense nucleic acidshybridize with the respective mRNA and form a double-stranded moleculewhich can then no longer be translated. The use of antisense nucleicacid is for example known from Marcus-Sekura, C. J., Anal. Biochem. 172(1988), p. 289-295.

Ribozymes are RNA molecules which are able to specifically cleave othersingle-stranded RNA molecules similar to DNA restriction endonucleases.The production of ribozymes is described in Cech, J. Amer. Med. Assn.260 (1988), p. 3030.

In this connection it is also possible according to the invention totransfect suitable vectors containing the DNA sequence according to theinvention in vitro or in vivo into patient cells or to transfect thevectors in vitro into cells and then to implant these in a patient.

MP121 antisense polynucleotides can also be introduced into cells whichexhibit an undesired expression of MP121.

The MP121 activity can also be suppressed by binding molecules to theMP121 receptors which, in contrast to MP121, do not trigger furthertransmission of a signal.

Thus within the scope of the invention the receptors for MP121 on cellsare also of interest. In order to find receptors, firstly various celllines can be tested for their binding properties with respect toradioactively labelled MP121 (¹²⁵I-MP121) with subsequent cross-linking.A cDNA library can subsequently be established in an expression vector(obtainable, from InVitrogen) from cells which bind MP121. Cells whichhave been transfected with receptor cDNA can then be selected by thebinding of radioactively labelled MP121. These are methods known to aperson skilled in the art and have for example been used to isolateactivin (Mathews, L. S. & Vale, W. W., Cell 65 (1991), 973-982) andTGF-β type II receptors (Lin, H. Y. et al., Cell 68 (1992), 775-785). Inanalogy to known activin receptors, the MP121 receptor is alsopresumably a receptor complex which belongs to this family so thatfurther methods known to a person skilled in the art, such as e.g. PCRwith degenerate oligonucleotides, can be used to find parts of theheteromeric complex. This method has also been used for example with theactivin and TGF-β type I receptors (Touchida et al., Proc. Natl. Acad.Sci. USA 90 (1993), 11242-11246; Attisano at al., Cell 75 (1993),671-680; Franzen et al., Cell 75 (1993), 681-692).

Finally the present invention concerns an antibody which can bindspecifically to the proteins according to the invention or such anantibody fragment (e.g. Fab or Fab′). Processes for the production ofsuch a specific antibody or antibody fragment are part of the generalknowledge of an average person skilled in the art. Such an antibody ispreferably a monoclonal antibody. Such antibodies or antibody fragmentscan also be suitable for diagnostic methods.

In addition it is intended to illustrate the invention by the followingexamples.

EXAMPLE 1

Isolation of MP121

1.1 Total RNA was isolated from human liver tissue (40 year old man)according to the method of Chirgwin et al. (Biochemistry, 18, 5294-5299(1979)). Poly (A+)-RNA was separated from the total RNA by oligo (dT)chromatography according to the manufacturer's instructions (Stratagenepoly (A) Quick columns).

1.2 For the reverse transcription reaction 1 to 2.5 μg poly (A+) RNA washeated for 5 minutes to 65° C. and quickly cooled on ice. The reactionmixture contained 27 U RNA-Guard (Pharmacia), 2.5 μg oligo. (dT)₁₂₋₁₈(Pharmacia), 5× buffer (250 mmol/l Tris/HCl pH 8.5, 50 mmol/l MgCl₂, 50mmol/l DTT, 5 mmol/l of each dNTP, 600 mmol/l KCl) and 20 U AMV reversetranscriptase (Boehringer Mannheim) per μg poly (A+) RNA. The reactionmixture (25 μl) was incubated for 2 hours at 42° C. The cDNA pool wasstored at −20° C.

1.3 The deoxynucleotide primers OD and OID shown in FIG. 2 were preparedon an automatic DNA synthesizer (Biosearch). Purification was carriedout by means of denaturing polyacrylamide gel electrophoresis andisolating the main bands from the gel by isotachophoresis. Theoligonucleotides were designed by comparing nucleic acid sequences ofknown members of the TGF-β family and selecting regions with highconservation. A comparison of this region is shown in FIG. 2. In orderto facilitate cloning, both oligonucleotides contained Eco RI cleavagesites and OD additionally contained a Nco I restriction cleavage site atits 5′ terminus.

1.4 In the PCR reaction cDNA corresponding to 20 ng poly (A+) RNA wereused as starting material (see 1.2) The reaction was carried out in avolume of 50 μl and contained 1×PCR buffer (16.6 mmol/l (NH₄)₂SO₄, 67mmol/l Tris/HCl pH 8.8, 2 mmol/l MgCl₂, 6.7 μmol/l EDTA, 10 mmol/lβ-mercaptoethanol, 170 μg/ml bovine serum albumin (Gibco), 200 μmol/l ofeach dNTP (Pharmacia), 30 pmol of each oligonucleotide (OD and OID) and1.5 U Taq polymerase, (AmpliTaq, Perkin Elmer Cetus). The reactionmixture was overlayed with paraffin and 40 PCR cycles were carried out.The products of the PCR reaction were purified by means ofphenol/chloroform extraction and concentrated by ethanol precipitation.

1.5 Half of the PCR reaction products was cleaved with the restrictionenzymes SphI (Pharmacia) and AlwNI (Biolabs) according to themanufacturer's instructions. The other half was cleaved in a series ofreactions using Ava I (BRL), AlwN I (Biolabs) and Tfi I (Biolabs). Therestrictions were carried out in 100 μl using 8 U enzyme for 2 to 12hours at 37° C. (apart from Tfi I at 65° C.).

1.6 The products of the restriction cleavage were fractionated by meansof agarose gel electrophoresis. After staining with ethidium bromide,uncleaved amplification products were cut out of the gel and isolated byphenol extraction. The DNA obtained was subsequently purified twice byphenol/chloroform extraction.

1.7 A quarter or a fifth of the isolated DNA was reamplified afterethanol precipitation using the same conditions as for the primaryamplification except that the number of cycles was reduced to 13. Thereamplification products were purified, cleaved with the same enzymes asabove and uncleaved products were isolated from the agarose gels aselucidated above for the amplification products. The reamplificationstep was repeated.

1.8 After the last isolation from the gel, the amplification productswere cleaved by 4 U Eco RI (Pharmacia) under the conditions recommendedby the manufacturer. A quarter of the restriction mixture was ligatedinto the vector pBluescript SK+ (Stratagene) which had been cleaved withEco RI. After ligation, 24 clones of each enzyme combination wereanalyzed further by sequencing. There were no new sequences in themixture which had been cleaved with AlwN I and Sph I, it contained onlyBMP6 and inhibin EA sequences. 19 identical new sequences, named MP121,were found in the mixtures cleaved with Ava I, AlwN I and Tfi I. Theseplasmids were named pSK-MP121 (OD/OID). One sequence differed by twonucleotides from this sequence that was otherwise found. Ligation andtransformation in E. coli was carried out as described in Sambrook etal., Molecular Cloning; A Laboratory Manual (1989).

The clone was extended to the 3′ and of the cDNA according to the methoddescribed in detail by Frohmann (published by Perkin-Elmer Corp.,Amplifications, 5, 11-15 (1990)). The same liver mRNA which had beenused to isolate the first MP121 fragment was reversely transcribed asdescribed above using oligo dT (16mer) linked to the adapter primer(AGAATTCGCATGCCATGGTCGACGAAGC-T₁₆). The amplification was carried outusing the adapter primer (AGAATTCGCATGCCATGGTCGACG) and an internalprimer (GGCTACGCCATGAACTTCTGCATA) prepared from the MP121 sequence. Theamplification products were prepared using a further internal primer(ACATAGCAGGCATGCCTGGTATTG) prepared from the MP121 sequence and with theadapter primer. After restriction with Sph I the reamplificationproducts were cloned into the vector pT7/T3 U19 (Pharmacia) which hadbeen cleaved in the same way and sequenced. The clones werecharacterized by their sequence overlap with the already known part ofthe MP121 sequence. One clone, named p121Lt 3′ MP13, was used to isolatea Nco I (made blunt using T4 polymerase)/Sph I fragment. This fragmentwas cloned into one of the above-mentioned pSK-MP121 (OD/OID) vectorswhose OD primer sequence was orientated towards the T7 primer of the pSKmultiple cloning site. For this the vector was cleaved with SphI andSmaI. The construct was named pMP121DFus6. It comprises the MP121sequence from position 922 to 1360 as shown in SEQ ID NO. 1.

1.9 A Dde I fragment of pMP121DFus6, which extends from position 931 to1304 in SEQ ID NO. 1, was used to screen a human liver cDNA library(Clontech, # HL3006b, lot 36223) as described in detail by Ausubel etal., (Current Protocols in Molecular Biology published by GreenePublishing Associates and Wiley-Interscience (1989)). 24 mixed plaqueswere picked from 8.1×10⁶ phages and separated. From this 10 clones whichyielded a positive signal using primer LO2 (ACATAGCAGGCATGCCTGGTATTG)and LOI1 (CTGCAGCTGTGTTGGCCTTGAGA) from the Dde I fragment were selectedanti separated. The cDNA was isolated from the phages by means of anEcoRI restriction and cloned into the pBluescript SK vector which hadalso been cleaved with EcoRI.

Sequencing of one of the resulting plasmids SK121L9.1 showed that thestart codon begins at position 128 of SEQ ID NO. 1 since three stopcodons are positioned in-frame in front of this start codon at positions62, 77 and 92. Mature MP121 starts at position 836 of SEQ ID NO. 1assuming sequence analogy to other TGF-β proteins which corresponds toamino acid 237 in SEQ NO. 2. The stop codon begins at position 1184 ofSEQ ID NO. 1.

Plasmid SK121L9.1 was deposited at the DSM on the 26.04.1994 under thedeposit number 9177.

1.10 Isolation of the MP121 cDNA and genomic DNA from the mouse: Thesequence information from the human MP121 sequence was used to isolatethe MP121 sequence from the mouse. The methods used for this are allknown to a person skilled in the art and are described for example inCurrent Protocols in Molecular Biology (Ausubel et al., GreenePublishing Associates and Wiley-Interscience, Wiley & Sons, 1987-1995)or in Molecular Cloning (Sambrook et al., second edition, Cold SpringHarbour Laboratory Press 1989). Firstly the primersACGAATTCCGACGAGGCATCGACTGC and GCGTCGACTACCATGTCAGGTATGTC derived fromthe human MP121 sequence containing additional restriction cleavagesites at the 5′ end (EcoR I or Sal I) were synthesized. These primerswere used for amplification on genomic mouse DNA. The 0.35 kb fragmentwhich results was subcloned in the Bluescipt vector (Stratagene) andused as a radioactive probe. A λ bank with genomic mouse DNA as well ana bank with cDNA was screened according to standard methods. The cDNAwas synthesized from RNA, which had been isolated from mouse liver andcloned into λgtl0 using EcoR I/Not I linkers. MP121 clones were isolatedfrom the genomic as well as from the cDNA bank. A cDNA containing thecomplete coding sequence was subcloned into the EcoR I cleavage site ofthe Bluescript vector SK (Stratagene) and the resulting plasmid SKMP121mouse was deposited on the 10.05.1995 at the DSM (DSM 996.4). Completesequencing resulted in the sequence shown in SEQ ID NO.3. The startcodon begins at position 131 in SEQ ID NO.3 and ends at the stop codonstarting at position 1187. The protein derived from the sequence isshown in SEQ ID NO.4. Subcloning and analyzing clones containing MP121from the genomic bank showed that the MP121 sequence contains an intronin the propeptide part of ca. 5.5 kb. This intron is located betweenpositions 446 and 447 in SEQ ID NO.3. The exon/intron junctions areshown in SEQ ID NO.5.

EXAMPLE 2

Expression of MP121

It is possible to express MP121 in eukaryotic as well as in prokaryoticsystems.

Only the mature part of MP121 was used for expression in prokaryotes.After purification the mature MP121 protein expressed in E. coli as amonomer can then be folded back to form a dimer. In order to simplifypurification of MP121, an additional 6 histidines can be attached to theN-terminus of the mature protein which facilitate purification of theprotein by binding to nickel-chelate columns.

As an example the mature part of human MP121 (amino acid 237 to 352 inSEQ ID NO.2) with an additional 13 amino acids, including 6 histidinesat the N-terminus, (MHHHHHHKLEFAM) was expressed in the prokaryoticvector pBP4. This vector is a pBR322 derivative having tetracyclinresistance which in addition contains the T7 promoter from thepBluescript II SK plasmid (Stratagene). Furthermore the vector containsa ribosomal binding site following the T7 promoter and a start codonfollowed by 6 codons for histidine. A terminator (TØ) follows afterseveral single restriction cleavage sites such as Eco RI, Xho I, Sma Iand Apa I for the insertion of inserts as well as stop codons in allthree reading frames. In order to obtain the cDNA for the mature part ofMP121, PCR was carried out on the plasmid SK121L9.1 (DSM depositarynumber: 9177) using the two oligonucleotidesGAATTCGCCATGGGCATCGACTGCCAAGGAGG and CCGCTGAGAAGCTTCAACTGCACCCACAGGC.Both oligonucleotides contain additional restriction cleavage sites attheir ends (Eco RI and Nco I or Xho I and Hind III). In an intermediatestep the resulting 377 bp fragment was cloned with blunt ends into thepBluescript II SK vector (Stratagene) that had been cleaved with Eco RV.One clone in the orientation of the 5′ end of MP121 towards the T7promoter was cleaved with Eco RI and the resulting insert (0.38 kb) wascloned into the pBP4 vector that had also been cleaved with Eco RI. Thecorrect orientation of the insert in the resulting plasmid pBP4MP121Hiswas established by restriction analysis and sequencing. The plasmidpBP4MP121His was deposited on the 30.1.1995 at the DSM (depositarynumber: 9704). The expression of MP121 protein can be achieved bysimultaneously providing T7 RNA polymerase. T7 RNA polymerase can beprovided by various methods such as e.g. by a second plasmid with a genefor T7 RNA polymerase or by infection with phages which code for T7 RNApolymerase or also by special bacterial strains which have integratedthe gene for T7 RNA polymerase. The nature MP121 protein with His-tag(MP121His) in produced in inclusion bodies by using the bacterial strainBL21 (DE3)pLysS (Novagen, #69451-1) and inducing the T7 RNA polymeraseexpression with IPTG according to the manufacturers instructions. In SDSpolyacrylamide gels (15%) the protein exhibits an apparent molecularweight of nearly 16 kD (theoretical molecular weight: 14.2 kD) as isshown representatively in the Western blot of FIG. 3. The bacteriatransformed with pBP4 as controls do not show any staining of specificbands. Due to the His-tag this protein can be purified onnickel-chelating agent columns as described for example by Hochuli etal., (BIO/Technology Vol. 6, 1321-1325 (1988)). An additionalpurification is possible by means of reversed phase HPLC. A reversedphase column (Nucleosil 300-7C4 from Macherey-Naget, Type 715023) wasused with a flow-rate of 2 ml/min and an acetonitrile gradient in 0.1%TFA of 0 to 90% within 100 minutes. MP121His elutes under theseconditions after ca. 40% acetonitrile.

The mature part of MP121 (amino acid 237 to 352 in SEQ ID NO.2) wasadditionally expressed in E. coli with one additional methionine at theN-terminus only using again a system with the T7 RNA Polymerase. Theexpression level was improved by including a gene for the lacI repressorin the expression plasmid (as it is used in the pET vectors fromNovagen) and using another E. coli strain, HMS 174 (DE3) (Novagen#69453). Inclusion bodies can be isolated by standard methods and washedwith 2 M guanidinium chloride/HCL in 20 mM Tris pH 8.0 MP121 is furtherpurified by a reversed phase HPLC as described for MP121His.

Additionally monoclonal antibodies were developed in mice. A peptide of26 amino acids from the mature part of MP121 was used as an antigen:PLSLLYYDRDSNIVKTDIPDMVVEAC. The antigen was coupled to ovalbumin usingthe free SH group of the cysteine according to conventional methods.Other constructs could be used as antigens also, as for example thedimeric mature MP121. Immunization of BALB/c mice was performedaccording to conventional methods. The coupled peptide was used forexample as antigen in combination with complete Freund's adjuvant forthe first immunization and in combination with incomplete Freund'sadjuvant in successive immunizations. The antigen (5-10 μg each time)was injected subcutaneously in the hind limbs of three mice at day 17,14, 10, 7, 4 and 1 before the isolation of popliteal lymphatic noneunderneath the knee joint. A suspension of cells was produced for fusionto myeloma cells (P3×63Ag8.653, ATCC, CRL 1580) by the help of PEG.These techniques are described in more detail by Peters, J. H. &Raumgarten, H. (1990, Monoklonale Antikörper—Herstellung undCharakterisierung, Springer Verlag, 2.Auflage). It is possible to selectfor fused hybridomas by addition of azaserine and hypoxanthine. Thesupernatants of different wells were tested after 8-10 days with ELISAand Western blot analyses using MP121 expressed in eukaryotic andprokaryotic cells. The cells with the best positive results were furthersubcloned to isolate cells producing only one monoclonal antibody. Topurify the monoclonal antibodies 1 liter of cell culture supernatantcontaining the monoclonal antibody was produced using roller bottles(Schott) in a “Cell-Roll” (Former Scientific) according to standardmethods.

The antibodies of the cell culture supernatants can be concentrated andsubsequently purified using a protein a column (ImmunoPure Plus (G) IgGPurification Kit, Pierce, #44795) according to the manufacturer'sinstructions. The monoclonal antibodies derived by this method proved tobe useful for the detection of MP121.

In each case the determination whether it is MP121 protein was carriedout by means of Western blot analysis using MP121-specific antibodies.Polyclonal antibodies against MP121 were produced in chicken as well asin rabbits. In order to obtain the antigen for the immunization, a partof the mature part of MP121 (amino acid 260 to 352 In SEQ ID NO.2) wasfused with the first 98 amino acids of the polymerase of the MS2bacteriophage and expressed in E. coli. After isolating the inclusionbodies, the fusion protein (MS2-MP121) was separated on polyacrylamidegels and isolated for the immunization after staining with copper bymeans of electro-alution (Tessmer, U. & Dernick, R., IBL (1990) 8-13).It is possible to specifically detect the expression of MP121 usingantibodies from chicken as well as from rabbits. Chicken antibodies wereused for the schematic Western blot in FIG. 3 which had been purifiedfurther by means of PEG precipitation (Thalley B. S. and Carroll, S. H.,BIO/Technology Vol. 8, 934-930 (1990)) and by means of membrane-boundantigen (fusion protein (MS2-MP121)) (18.17 in Sambrook at al.,Molecular Cloning, second edition, Cold Spring Harbor Laboratory press1989). Anti-chicken IgG coupled to alkaline phosphatase (Sigma A9171)was used as the second antibody. The detection was carried out accordingto the manufacturer's instructions using the Tropix Western-LightProtein Detection Kit (Serva #WL10RC).

In order to obtain biologically active material, the purified monomericMP121 expressed in E. coli can be folded back to form a dimeric MP121.This can be carried out according to the methods for example describedby Jaenicke, R. & Rudolph, R. (Protein structure, ed. Creighton, T. E.,IRL Press, chapter 9).

The Vaccinia virus expression system was, used for expression ineukaryotic cells as it is described in detail and in a form which caneasily be repeated by a person skilled in the art in Current Protocolsin Molecular Biology (Ausubel et al., Greene Publishing Associates andWiley-Interscience, Wily & Sons) abbreviated in the following as CP, inchapter 16 unit 16.15-16.18. The system is based on the fact thatforeign DNA can be integrated by homologous recombination into thegenome of the Vaccinia virus using certain vectors. For this purpose thevector used contains the TK (thymidine kinase) gene from the Vacciniagenome. In order to enable selection for recombinant viruses, the vectoradditionally contains the E. coli xanthine-guanine-phosphoribosyltransferase gene (gpt) (Falkner, F. G. & Moss, B., J. of Virol. 62(1900), 1049-1854). The cDNA with the complete region coding for MP121was cloned into this vector.

PCR reactions and intermediate cloning was necessary in order to shortenthe 5′ and 3′ untranslated regions of the initial plasmid SK121L9.1(DSM, depositary number: 9177) and to insert single restriction cleavagesites at the ends. All PCR reactions were carried out using the plasmidSK121L9.1 (DSM despositary number: 9177). In order to shorten the 5′untranslated end, the primer CCCGGATCCGCTAGCACCATGACCTCCTCATTGCTTCTGwith an inserted Bam HI and NheI restriction cleavage site was used in aPCR with an internal primer (CCCTGTTGTCCTCTAGAAGTG). In an intermediatestep the fragment obtained was cloned into Bluescript. SK (Stratagene),sequenced and checked for concurrence with the sequence shown in SEQ IDNO.1. The Sph I/Eco RI fragment (0.22 kb) from the plasmid pBP4MP121Hiswas used to shorten the 3′ untranslated end.

Both end fragments of MP121 were linked to the missing middle DNAsequence from the plasmid SK121L9.1 (DSM depositary number: 9177) bymeans of internal restriction cleavage sites (Xba I and Sph I) accordingto standard methods (Sambrook et al. Molecular Cloning, second edition,Cold Spring Harbor Laboratory Press 1989). The shortened cDNA obtainedin this way having the complete reading frame for MP121 (nucleotide 128to nucleotide 1184 in SEQ ID NO.1) could be cloned into the vector pBP1which had also been cleaved by using the restriction cuts Bam HI and EcoRI. The resulting plasmid pBP1MP121 was deposited on 12.1.95 at the DSM(depositary number: 9665).

The plasmid pBP1MP121 was used for the production of recombinantVaccinia viruses. For this 143B cells (HuTk-, ATCC CRL 8303) which were80% confluent were infected with vaccinia wild-type virus (1 virus per10 cells) in 1 ml PBS in 35 ml culture plates for 30 minutes at roomtemperature while shaking occasionally. After aspirating the supernatantand adding 2 ml culture medium (MEM, Gibco BRL #041-01095 containing1,500 diluted penicillin and streptomycin Gibco BRL #043-05140), theywere incubated for 2 hours at 37° C. Subsequently the medium was removedand these cells were transformed for ca. 15 hours; at 37° C. using 100ng pBP1MP121, 2 μg carrier DNA (calf thymus, treated with ultrasound,Boehringer Mannheim #104175) and 10 μl Lipofectin (Gibco BRL #18292-011)in 1 ml MEM. After addition of 1 ml MEM containing 20% FCS (Sigma#F-7524) they were incubated for a further 24 hours at 37° C. andsubsequently the lysed cells were frozen.

Gpt selection for xanthine-guanine-phosphoribosyl transferase andisolation and amplification of individual recombinant viruses wasessentially carried out as described in unit 16.17 of CP with thedifference that RK13 cells (ATCC CCL 37) wore used.

Integration of the MP121 cDNA into the viral genome was confirmed by dotblot analysis (CP unit 16.18). A recombinant virus from the transfectionwith pBPMP121 and the wild-type virus were used for expression analysedin cell lines 143B (HuTk-, ATCC CRL 8303, human) and NIH-3T3 (DSM ACC59, Swiss mouse embryo). The cells were cultured according to thedistributor's instructions. Confluent cells were infected for 30 minutesat 37° C. with the three-fold number of viruses and subsequently therespective culture medium containing 10% FCS and penicillin/streptomycin(1:500, Gibco BRL #043-05140) was added. The medium was removed after 6hours at 37° C., the cells were washed twice with e.g. HBSS (Gibco BRL∩14180-046) and production medium (MEM for HuTk- or DMEM containing 4.5g/l glucose and NEAA (Gibco BRL #11140-035) for NIH-3T3 each of whichcontained aprotinin (Fluka #10820, 50 U/ml) and penicillin/streptomycin)without FCS. After a production period of 20 to 22 hours, the cellsupernatant was collected. The expression was analysed by weans ofWestern blots according to standard methods (CP unit 10.8). For this theproteins from 1 to 3 ml cell culture supernatant were precipitated byaddition of an equivalent volume of acetone and incubating for at leastone hour on ice and centrifuged. After resuspending the pellets inapplication buffer (7 M urea, 1% SDS, 7 mM sodium dihydrogen phosphate,0.01% bromophenol blue and 1% β-mercaptoethanol if desired) they wereseparated in 15% polyacrylamide gels. A pre-stained protein molecularweight standard (Gibco, BRL #6041-020) was used as marker proteins.Transfer onto a PVDF membrane (Immobilon #IPVH00010) and blocking themembrane was carried out according to standard methods.

A representative schematic diagram of the results of the Western blot inFIG. 3 shows that MP121-specific bands occur in the recombinant virusinfected cells. The expression of MP121 in NIH-3T3 cells leads to asecreted protein with an apparent molecular weight in the gel of about18 kD under non-reducing conditions (expected theoretical molecularweight: 25 kD). Under reducing conditions the protein migrates at about15 kD in the gel (expected theoretical molecular weight: 12.5 kD). Theseresults show that MP121 is expressed as a dimeric mature protein asexpected. The migration behaviour of the dimeric MP121 protein which isonly slightly slower than the monomeric MP121 protein is probably due toits globular structure. The processing of the precursor protein to formthe mature protein could also be demonstrated in HuTk cells. No bandsoccurred in the Western blot with cells (HuTk- or NIH-3T3) infected withwild-type viruses (without integrated foreign DNA).

Further expression studies of MP121 using the Vaccinia virus systemrevealed that several cell lines express in addition to the dimericMP121 also significant amounts of a monomeric form. This monomeric formseems to be folded and has a more globular structure because it runsfaster in PAGE/Western blot analyses than the reduced monomer derivedfrom the dimeric MP121 after treatment with DTT. FIG. 6 shown theexpression of dimeric and monomeric MP121 in HepG2 cells (Hepatocellularcarcinoma, human, ATCC HB 8065). A residual unprocessed precursor formappears in addition. It was already shown by our Northern blot analysisthat the HepG2 cells naturally transcribe the MP121 gene, therefore itcan be assumed that the appearance of monomeric MP121 is ofphysiological relevance. The monomeric MP121 was found besides thedimeric MP121 in significant amounts in Mv1Lu (NBL-7, lung, mink, ATCCCCL 64) and Hela (Epitheloid carcinoma, cervix, human, ATCC CCL2) too.In addition, MP121 was expressed using the Baculovirus expression system(Invitrogen). After infection of insect larvae (Trichoplusia ni) withrecombinant viruses, MP121 was detected in the haemolymph after 3-4 daysin the dimeric form.

When co-transfection with recombinant vaccinia viruses that code forvarious members of the TGF-β family has also taken place, the vacciniavirus expression system is also particularly suitable for the productionof heterodimers. It is then possible to separate heterodimers fromhomodimers by affinity columns using specific antibodies against theindividual members of the TGF-β family. In this case the a as well as βAand βB chains of inhibins are of particular interest.

EXAMPLE 3

Investigation of the Expression of MP121 in Various Mouse Tissues

Total RNA from various tissues (brain, heart, kidney, liver, lung,spleen, muscle, ovary, testes) was isolated according to standardmethods from 6 week-old mice as well as from embryonic stem cells. 10 μgtotal RNA was used in each case in a RNAse protection assay (RPA) fromAmbion (RPA II kit, #1410) according to the manufacturer's instructions.In order to obtain specific probes for activin β_(A) and activin β_(B)the genomic DNA from the mouse (129Sv) was amplified from the maturepart, of the proteins using corresponding specific primers. In order tofacilitate cloning, EcoR I and/or BamH I or Hind III restrictioncleavage sites were introduced respectively at the ends of the primers.In the case of activin β_(A) the primers were derived from mRNA fromrats (GenBank Accession #M37482): GGATCCGAATTCGGCTTGGAGTGTATGGCAAGG andGGATCCGAATTCCTCTGGGACCTGGCAACTCTAG.

In the case of activin β_(B) degenerate primers were derived from thehuman sequence (Mason et al., Molecular Endocrinology 3, 1352-1358(1989): GAGAATTCCA (GA) CA (GA) TT (TC) TT (CT) AT and GCAAGCTTT (GA) TA(TC) TC (GA) TC (GA) TC.The resulting PCR fragments were subcloned into the vector pGEM-4(Promega) and tested. The activin-specific and thus in the RPA protectedsequences have a fragment size of 369 bp in the case of activin β_(A)and 254 bp in the case of activin β_(B). In MP121 the protected fragmentcomprises the sequence from position 887 to position 1164 in SEQ IDNO.3. The fragments cloned into pGEM-4 were transcribed in vitro inorder to produce radioactively labelled antisense RNA probes. This wascarried out according to the manufacturer's instructions (Promega,Riboprobe Gemini Systems) using 100 μM CTP and at the same time α³²P-CTP(800 Ci/mmol, Amersham). A radioactively labelled RNA was alsosynthesized as a control from the plasmid pTri-GAPDH (Ambion #7431)linearized with Dde I but using 1 mM CTP. After isolating the 4antisense RNA probes from polyacrylamide gels, these were incubated at42° C. overnight in the same mixture with the respective tissue RNA fromthe mouse (10 μg total RNA per probe having 1×10⁵ cpm). It was analyzedin a denaturing gel according to standard methods with a subsequentautoradiography for 4 days.

The analysis of MP121 mRNA expression in liver cells or remnant liverwas performed likewise or using Northern blot analysis according tostandard procedures (see CP, Chapter 4 or Molecular Cloning, Sambrook etal., 2nd Edition, Cold Spring Harbor Laboratory Press 1989). Hepatocyteswere isolated from rat (Wistar) liver and cultured according to Yasudaet al. (J. Clin. Invest. Vol. 92, 1491-1496 (1993)). The cells werewashed prior to incubation with fresh serum-free medium containing 0.1nM insulin. 0.1% BSA, optionally 1 nM EGF and (1 nM) partially purifiedMP121 (see Example 4). Partial hepatectomy (about 70% of the rat liver)was performed as described by Higgins & Anderson (Arch. Pathol. 12,186-202 (1931)) under ether anesthesia.

EXAMPLE 4

Partial Purification of MP121 and Examination of the Activity ofPartially Purified MP121

The MP121 protein which had been obtained by expression in the Vacciniavirus system (see example 2) could be partially purified by means of twocolumns.

In order to produce MP121 confluent NIH-3T3 cells (DSM ACC 59, Swissmouse embryo) were infected with the same number of recombinant virusesfor 30 minutes at 37° C. and subsequently the appropriate culture mediumcontaining 10% FCS and penicillin/streptomycin was added.

After 4 hours at 37° C. the medium was removed, the cells were washedtwice and production medium (see Example 2) without FCS was added. After20 to 22 hours production, the cell supernatant was collected andcentrifuged in order to remove the viruses (40000×g for 30 minutes at 4°C.) and filtered (0.1 μm pore size, Millex VV, Millipore #SLVV25LS). Thecontrol supernatant (wt) was obtained in a comparable manner afterinfection by wild-type Vaccinia viruses. The expression of MP121 waschecked by means of Western blot analysis and estimated to be 50-100μg/l.

The cell culture supernatant containing MP121 (1.1 l) was admixed withthe protease inhibitor PMSF (1 μM) brought to a final concentration of 1M (NH₄)₂SO₄, 20 mM Tris pH 8.0 and loaded onto a phenyl-Sepharose (fastflow (high sub) Pharmacia #17-0973-05) column (5 ml bed) equilibrated inbuffer A (1 M (NH₄)₂SO₄, 20 mM Tris pH 8.0). The loaded column waswashed with 15 column volumes of buffer A and 10 column volumes ofbuffer B (20 mM Tris pH 8.0) and eluted within 50 minutes (5 ml perfraction) with a linear gradient to 100% buffer C (20 mM Tris pH 8.0,80% ethylene glycol) at a flow rate of 1 ml/min. It was possible tocheck that MP121 eluted between 50 and 80% ethylene glycol by means ofWestern blot analysis. Aliquots of these fractions were examined using15% polyacrylamide silver-stained gels according to the manufacturer'sinstructions (Silver Stain-II, Daiichi #SE140000) and the fractionscontaining MP121 were pooled. After purification of the controlsupernatant comparable fractions were also pooled after analysis insilver-stained gels.

The pooled fractions were purified further with the aid of reversedphase HPLC. For this a C8 column (Aquapore RP300, Applied Biosystems,particle size; 7 μm, pore size; 300 Å) was equilibrated with buffer A(0.1% trifluoroacetic acid/water). After loading the column with thepooled fractions containing MP121 from the phenyl-Sepharose column, itwas extensively washed with buffer A. The bound protein was eluted at aflow rate of 0.2 ml/min using a linear gradient of 1.5% buffer a (90%acetonitrile, 0.1% trifluoroacetic acid) per minute. Fractions of 600 μlwere collected and analyzed in a Western blot as well as withsilver-stained gels. Under the selected conditions MP121 protein elutedafter about 55% acetonitrile. The fractions containing MP121 werepooled. The same was carried out with the corresponding fractions fromthe purification of the control supernatant. The analysis in the silvergel showed that MP121 was still contaminated by other proteins. Furtherpurification steps are necessary to obtain pure MP121.

Other methods known to a person skilled in the art such as gel sievecolumns, ion exchange columns, affinity columns or metal chelate columnscould also be used for the further purification.

It was estimated from Western blot analysis that ca. 8 μg partiallypurified MP121 was obtained from 1 l of cell culture supernatant. Thepartially purified protein was stored lyophilized at −80° C.

In order to investigate the influence of MP121 on dopaminergic neurones,neurones from the mesencephalic floor of 14 day-old rat embryos (E14)were isolated according to a method described by Shimoda et al. (BrainRes. 586, 319-331 (1992)). The cells were singled out and cultured asdescribed by Krieglstein et al., (Neuroscience 63, 1189-1196 (1994)).The cell density on polyornithine/laminin-coated cover glasses is 200000cello/cm². After culture for 24 hours and subsequently every three daystwo-thirds of the medium (500 μl) was removed and replaced by freshmedium containing the respective additives. The lyophilized MP121partially purified by phenyl sepharose and reversed phase HPLC wasdissolved in 50% acetonitrile and added to the medium. The finalconcentration of MP121 in the medium is 20 ng/ml (the finalconcentration of acetonitrile is 0.3%). A comparable amount from thecontrol supernatant (wt) which had been purified in a comparable mannerwas dissolved in 50% acetonitrile and added. The medium control alsocontains 0.3% acetonitrile. After eight days the cultures were fixed for10 minutes at room temperature in 4% paraformaldehyde; the cells weremade permeable with acetone (10 min, −20° C.) and washed with PBS(phosphate buffered saline). After treatment with 1% H₂O₂ in PBS,washing and blocking with horse serum, they were stainedimmunocytochemically. Tyrosine hydroxylase (TH) is a limiting enzyme inthe biosynthesis of dopamine and other catecholamines so that TH can beused as a marker for dopaminergic neurones in the present cultures(cells containing noradrenaline were not isolated). TH was detected by a1 hour incubation at 37° C. using a mouse-monoclonal antibody againstrat TH (diluted 1:200, Boehringer Mannheim) and subsequent detectionusing the Vectastair ABC kit (Vecto Labs). TH-positive cells werecounted in an area of 0.12 cm². It can be seen from FIG. 5 that MP121has a positive effect on the survival of dopaminergic neurones.

In order to investigate the neural influence of MP121 in another systemexplant cultures of the embryonic retina were used. This organotypicculture system is described in detail by Carri, N. G. & Ebendal, T.(Dev. Brain Res. 6, 219-229 (1983)), Carri, N. G. & Ebendal, T. (Anat.Rec. 214, 226-229 (1956) and Carri, N. G. et al. (J. Neurosci. Res. 19,428-439 (1988)). This assay measures the stimulation of extending nervefibres from the embryonic retina on a collagen substratum. Briefly, theretinal explants were taken from the chick retina (White Leghorn,embryonic day 6) and the neural retina was separated from the pigmentepithelium and mesenchymal cells by repeated washing. The organotypicexplants were transferred to collagen-coated culture dishes andincubated overnight (37.5° C., 5% CO₂). The lyophilized MP121 partiallypurified by Phenyl-Sepharose and reversed phase HPLC was dissolved inaqueous buffer or 50% acetonitrile and diluted in the culture medium toa final concentration of 1.25 ng/ml, 12.5 ng/ml, 25 ng/ml, 50 ng/ml, 100ng/ml, 200 ng/ml, whereby it makes no difference in the results whetheracetonitrile or aqueous buffers were used for solubilization. Acomparable amount from the control supernatant (wt) which had beenpurified in a comparable manner was added in control assays. For thebackground fibre outgrowth, standard tissue culture medium with onlybovine serum added was used. The incubation was continued and after a 4day period in culture the maximum length of the leading fascicles wasmeasured in an inverted microscope under dark-field illumination. Asshown in Table 1, MP121 dose-dependently stimulated the outgrowth ofnerve fibres being maximally active at about 25 ng/ml resulting in areal fibre length of about 1.7 mm. FIG. 7 shows the fibre outgrowth in aliving culture after treatment with MP121 (5 ng/ml). The control (wt)did not stimulate fibre outgrowth as tested in concentrations equivalentto those used for the active MP121. TABLE 1 Retinal neurite length after4 days in culture treated with different concentrations of MP121. Theneurite lengths of the background fibre outgrowth in the control tissueculture medium were 5.5/8/10/11/4.8/7 units giving a mean of 7.7 units(SEM 1.00). The neurite lengths of the wt control (used in equivalentconcentrations as MP121) was in the same range as the background fibres.Each unit represents 0.03 mm real scale in the culture dish. MP121(ng/ml) Length (units) Mean ± SEM 1.25 7/12/5/6  7.5 ± 1.5 12.519/20/13/26 19.5 ± 2.6 25 50/52/60/71/65/53 58.5 ± 3.4 5037/32/48/41/36/20 35.6 ± 3.8 100 21/8/19/18 16.5 ± 2.9 200 11/8/12/1010.2 ± 0.8

In order to investigate the influence of MP121 on liver derived cellshepatocytes were isolated from rat (Wistar) liver and cultured accordingto Yasuda et al. (J. Clin. Invest. Vol. 92, 1491-1496 (1993)). The cellswere washed prior to incubation with fresh serum-free medium containing0.1 nM insulin, 0.1% BSA and 1 nM BOP. The lyophilized MP121 partiallypurified by phenyl sepharose and reversed phase HPLC was solubilized inacetonitrile as usually and added to the medium at variousconcentrations (see FIG. 8). A comparable amount from the controlsupernatant (wt) which had been purified in a comparable manner was usedas a control. The hepatocytes were incubated for 72h and 0.5 μCi[³H]Thymidine/ml was included for the last 24 hours au described by Mead& Pausto (Proc. Natl. Acad. Sci. USA 86, 1558-1562 (1989)).[³H]Thymidine incorporation into trichloracetic acid-perceptablematerial was subsequently measure as described by McNiel et al. (J. CellBiol. 101, 372-379 (1985)).

In order to investigate the influence of MP121 on erythroiddifferentiation its influence on Friend leukemia cells (F5-5) wasmeasured. Therefore Friend leukemia cells were cultured in microliterplates essentially as described by Eto et al. (Biochem. Biophys. Res.Com. 142, 1095-1103 (1987)). The lyophilized MP121 partially purified byphenyl sepharose and reversed phase HPLC was solubilized as alreadydescribed, added to the Friend cells at various concentrations (see FIG.9) and incubated for 5 days. The percentage of differentiated cells wasdetermined after staining with o-dianicidine.

1. An isolated antibody or antibody fragment which specifically binds toa protein of the TGF-β family which has mitogenic and/ordifferentiation-inductive activity, wherein the antibody or antibodyfragment binds to a part of the protein encoded by a nucleotide sequenceselected from the group consisting of: (a) the nucleotide sequence shownin the SEQ ID NO:1, or the following fragments: nucleotides 128-1183 ofSEQ ID NO:1, nucleotides 836-1183 of SEQ ID NO:1, and nucleotides866-1183 of SEQ ID NO:1; (b) the nucleotide sequence shown in the SEQ IDNO:3, or the following fragments: nucleotides 131-1186 of SEQ ID NO:3,nucleotides 839-1186 of SEQ ID NO:3, and nucleotides 869-1186 of SEQ IDNO:3; (c) a nucleotide sequence encoding the amino acid sequence encodedby (a) or (b); and (d) a DNA molecule comprising a nucleotide sequence(i) which hybridizes with a complement of one of the DNA molecules from(a) and (b) under stringent hybridization conditions in 6×SSC at 62-66°C. followed by one hour wash with 0.6×SSC and 0.1% SDS at 62-66° C. and(ii) which encodes a protein comprising an amino acid sequencecontaining seven conserved cysteine residues, said seven conservedcysteines corresponding to cysteine residues at positions 247, 276, 280,316, 317, 349 and 351 in SEQ ID NO:2.
 2. The isolated antibody orantibody fragment of claim 1, wherein the antibody is a monoclonalantibody.
 3. The isolated antibody or antibody fragment of claim 1,wherein the antibody or antibody fragment specifically binds to a partof the protein selected from the group consisting of: (a) a proteincomprising an amino acid sequence set forth in SEQ ID NO:2; (b) aprotein comprising an amino acid sequence set forth in SEQ ID NO:4; (c)a mature protein that starts with one of amino acids 217-240 and endswith amino acid 352 of SEQ ID NO:2; (d) a mature protein which comprisesat least the region of seven cysteine residues, said region comprisingamino acid residues 247-352 of SEQ ID NO:2; (e) a mature protein thatstarts with one of amino acids 217-240 and ends with amino acid 352 ofSEQ ID NO:4; and (f) a mature protein which comprises at least theregion of seven cysteine residues, said region comprising amino acidresidues 247-352 of SEQ ID NO:4.
 4. The isolated antibody or antibodyfragment of claim 3, wherein the antibody is a monoclonal antibody. 5.The isolated antibody or antibody fragment of claim 1, wherein theprotein is selected from the group consisting of: (a) a mature proteinthat starts with amino acid 236 and ends with amino acid 352 of SEQ IDNO:2; (b) a mature protein that starts with amino acid 237 and ends withamino acid 352 of SEQ ID NO:2; (c) a mature protein that starts withamino acid 236 and ends with amino acid 352 of SEQ ID NO:4; (d) a matureprotein that starts with amino acid 237 and ends with amino acid 352 ofSEQ ID NO:4;
 6. The isolated antibody or antibody fragment of claim 5,wherein the antibody is a monoclonal antibody.
 7. A kit for detecting aprotein of the TGF-β family comprising (a) the antibody or antibodyfragment of claim 1 and (b) a reaction buffer.
 8. A kit for detecting aprotein of the TGF-β family comprising (a) the antibody or antibodyfragment of claim 2 and (b) a reaction buffer.
 9. A kit for detecting aprotein of the TGF-β family comprising (a) the antibody or antibodyfragment of claim 3 and (b) a reaction buffer.
 10. A kit for detecting aprotein of the TGF-β family comprising (a) the antibody or antibodyfragment of claim 4 and (b) a reaction buffer.
 11. A kit for detecting aprotein of the TGF-β family comprising (a) the antibody or antibodyfragment of claim 5 and (b) a reaction buffer.
 12. A kit for detecting aprotein of the TGF-β family comprising (a) the antibody or antibodyfragment of claim 6 and (b) a reaction buffer.
 13. A method fordetecting a protein of the TGF-β family comprising (a) incubating theantibody or antibody fragment of claim 1 with a sample suspected ofcontaining said protein and (b) detecting any antibody/protein complexformed, wherein the presence of the complex indicates the presence ofthe protein.
 14. A method for detecting a protein of the TGF-β familycomprising (a) incubating the antibody or antibody fragment of claim 2with a sample suspected of containing said protein and (b) detecting anyantibody/protein complex formed, wherein the presence of the complexindicates the presence of the protein.
 15. A method for detecting aprotein of the TGF-β family comprising (a) incubating the antibody orantibody fragment of claim 3 with a sample suspected of containing saidprotein and (b) detecting any antibody/protein complex formed, whereinthe presence of the complex indicates the presence of the protein.
 16. Amethod for detecting a protein of the TGF-β family comprising (a)incubating the antibody or antibody fragment of claim 4 with a samplesuspected of containing said protein and (b) detecting anyantibody/protein complex formed, wherein the presence of the complexindicates the presence of the protein.
 17. A method for detecting aprotein of the TGF-β family comprising (a) incubating the antibody orantibody fragment of claim 5 with a sample suspected of containing saidprotein and (b) detecting any antibody/protein complex formed, whereinthe presence of the complex indicates the presence of the protein.
 18. Amethod for detecting a protein of the TGF-β family comprising (a)incubating the antibody or antibody fragment of claim 6 with a samplesuspected of containing said protein and (b) detecting anyantibody/protein complex formed, wherein the presence of the complexindicates the presence of the protein.
 19. A method according to claim13 for diagnosing a disease which is associated with the expression ofMP121.
 20. A method according to claim 14 for diagnosing a disease whichis associated with the expression of MP121.
 21. A method according toclaim 15 for diagnosing a disease which is associated with theexpression of MP121.
 22. A method according to claim 16 for diagnosing adisease which is associated with the expression of MP121.
 23. A methodaccording to claim 17 for diagnosing a disease which is associated withthe expression of MP121.
 24. A method according to claim 18 fordiagnosing a disease which is associated with the expression of MP121.25. A method for suppressing the mitogenic and/ordifferentiation-inductive activity of a protein of the TGF-β familycomprising incubating the antibody or antibody fragment of claim 1 withsaid protein, wherein said protein is inactive in the antibody/proteincomplex formed.
 26. A method for suppressing the mitogenic and/ordifferentiation-inductive activity of a protein of the TGF-β familycomprising incubating the antibody or antibody fragment of claim 2 withsaid protein, wherein said protein is inactive in the antibody/proteincomplex formed.
 27. A method for suppressing the mitogenic and/ordifferentiation-inductive activity of a protein of the TGF-β familycomprising incubating the antibody or antibody fragment of claim 3 withsaid protein, wherein said protein is inactive in the antibody/proteincomplex formed.
 28. A method for suppressing the mitogenic and/ordifferentiation-inductive activity of a protein of the TGF-β familycomprising incubating the antibody or antibody fragment of claim 4 withsaid protein, wherein said protein is inactive in the antibody/proteincomplex formed.
 29. A method for suppressing the mitogenic and/ordifferentiation-inductive activity of a protein of the TGF-β familycomprising incubating the antibody or antibody fragment of claim 5 withsaid protein, wherein said protein is inactive in the antibody/proteincomplex formed.
 30. A method for suppressing the mitogenic and/ordifferentiation-inductive activity of a protein of the TGF-β familycomprising incubating the antibody or antibody fragment of claim 6 withsaid protein, wherein said protein is inactive in the antibody/proteincomplex formed.